In Situ Hybridization
for
Light and Electron Microscopy
Using
Ultra Small Immunogold
Detection
and
Silver Enhancement

Newsletter 3

Aim of this Newsletter

Whereas immunocytochemistry (IC) mainly deals with
the localization of specific proteins, in situ
hybridization (ISH) aims at the localization of specific
DNA or RNA sequences. Both approaches require the
preservation and exposure of target molecules in
biological specimens as well as the preservation of
ultrastructural details to allow high resolution detection.
Since between the two approaches target molecule
chemical characteristics differ significantly, specimen
preparation conditions as well as specific detection
procedures will have to be adapted accordingly.

In this Newsletter procedures are presented that allow
the sensitive intranuclear and cytoplasmic detection of
28S ribosomal RNA (rRNA) by ISH as a model system.
To this end a specimen preparation technique was
adapted to allow preservation and exposure of RNA.
Secondly, a pre-embedding immunogold detection
procedure is presented which warrants a high degree of
penetration and detection efficiency. As a derivative a
post-embedding procedure is presented. Apprehensive
schedules and extensive procedures illustrate the
specimen preparation techniques used, as well as the in
situ hybridization and the pre and post-embedding
labeling protocols.

Although 28S rRNA is quite abundant the high label
density and structural integrity provide a firm basis for
the sensitive detection of low copy numbers of both
RNA and DNA sequences.

Introduction

An extensive number of biochemical studies deal with
nuclear functions like replication of DNA, transcription
and metabolism of mRNA. However relatively little is
known about the localization of these nuclear functions.
In situ hybridization, a detection technique which
enables the localization of specific nucleic acid
sequences, will be of help to place the nuclear functions
in a morphological context. Especially when applied at
the electron microscopical level, in situ hybridization
enables the correlation of for instance specific RNA
sequences with cytoplasmic and nuclear structures like
the cytoskeleton, interchromatin granules, nucleolar
sub-structures, lamines and the nuclear matrix.

The nuclear matrix is defined as the structure which
remains after removal of most of the DNA with DNAse
and high salt. The structure is thought to play an
essential role in nuclear functions (reviewed in
Verheyen et al 1988) and is a topic of research at the
Molecular Cell Biology Department. Localization of
specific RNA sequences in such preparations may
elucidate the involvement of the nuclear matrix in
nuclear functions.

Gold particles are the marker of choice for the electron
microscopical detection of target molecules. The use of
ultra small gold particles, the smallest available, in
combination with silver enhancement results in
maximized labeling efficiency (De Graaf 1991; Humbel
and Biegelmann 1992) and the possibility to evaluate
the results both at the light and electron microscopical
level.

The combined in situ hybridization and immunogold
detection procedures can be performed in the pre and
post-embedding mode.

Post-embedding in situ hybridization/immunogold
labeling (ISH/IG) is performed on sections. The
specimen preparation steps (fixation, dehydration and
embedding) are critical for the adequate preservation of
the target sequences. As opposed to pre-embedding
approaches it enables the localization of different
nucleic acid sequences in serial sections.

Pre-embedding ISH/IG is performed on fixed but
unembedded cells or tissue. It is only feasible when the
target nucleic acid sequences are within reach of both
the nucleic acid probe and the immunogold reagent.
Limited accessibility makes it necessary to apply
detergents (see for an extensive description of preembedding
immunocytochemical localization using
ultra small gold conjugates the AURION Newsletter 5).

It has already been shown that nuclear proteins can be
successfully localized in nuclear matrix preparations,
using pre-embedding immunolabeling with ultra small
gold conjugates and silver enhancement (de Graaf
1992). We have combined this pre-embedding labeling
technique with in situ hybridization experiments for the
localization of specific RNA sequences in the nuclear
matrix. The pre-embedding in situ hybridization
approach was developed since pre-embedding labeling
of permeabilized specimens results in in-depth labeling
throughout the whole cell or nuclear matrix, whereas
with post-embedding labeling only those targets can be
reached which are exposed at the surface of resin
sections. Using this approach EGF-receptor transcripts
have been successfully localized in nuclear matrices
(submitted for publication).

With the pre-embedding approach we localized 28S
rRNA using a digoxigeninylated DNA probe specific
for human 28S rRNA and a F(ab) anti-digoxigenin
Ultra Small ImmunoGold conjugate. The 28S rRNA
was detected in the nucleolus, a dense and relatively
difficultly accessible nuclear structure.

In this Newsletter a detailed protocol is presented for
the use of Aurion ultra small gold conjugates in the preembedding
in situ hybridization study as mentioned
above. It is demonstrated that with this labeling method,
hybridization and detection methods can be optimized
first at the light microscopical level before proceeding
with electron microscopy.

The light microscopical detection obtained with the
immunogold conjugate is related to the detection using
alkaline phosphatase as a marker.

Whereas most steps are explained at length in the "Detailed Procedure", the missing steps are
explained below.

Pre-treatment

Pre-treatment steps can be used in both pre and post-embedding in situ hybridization methods to improve the signal by
digesting proteins surrounding the target nucleic acid sequences. These steps involve an incubation with e.g.
proteinase K (up to 500 µg/ml in 200 mM Tris-HCl, 2 mM CaCl2, pH 7.4 for 5-30 minutes at 37°C) or with pepsin
(500 µg/ml in 0.2 M HCl at 37°C for 5-30 minutes) prior to the hybridization. However, since these treatments affect
the ultrastructure, a compromise has to be found between optimal signal and acceptable morphological preservation.

Pre-Hybridization

To improve the hybridization signal/noise ratio a pre-hybridization step can be introduced using the hybridization
mixture with omission of the nucleic acid probe. The presumed action is two-fold:

by melting out the hairpins present in the RNA sequence under investigation at higher temperatures (50-80°C) more
target sequences are exposed,

) background on accord of aspecific nucleic acid interactions is blocked by the herring sperm DNA and yeast tRNA
present in the hybridization mix.

In the present study the application of pre-treatment or pre-hybridization steps did not result in signal improvement. Therefore these steps were no longer included in the presented protocol.

Detailed Procedure for Pre-embedding ISH/IG

Permeabilization and Fixation

A431 cells were grown on coverslips for light microscopy
and on thermanox for electron microscopy.
Nuclear matrix preparations were made according to Fey et al (1986) as follows:
All steps are at room temperature, unless indicated otherwise.
Remove specimens from the growth medium, transfer to wells filled with RNAse free PBS.
Remove PBS immediately and extract the cells on ice for 3 minutes in CSK-buffer.
Digest the chromatin with 1000 U/ml DNase I in CSK buffer for 30 minutes,
Wash in 250 mM ammonium sulphate (RNAse-free) to remove DNA digests.

Discussion and Evaluation

In this newsletter a detailed protocol for the light and
electron microscopical detection of nucleic acid
sequences is presented. The use of the immunogold
silver detection system offers the possibility to work out
protocols at the LM level and to extend the approach to
EM specimens.

Ultra small gold particles are shown to be suited for the
sensitive pre-embedding in situ hybridization at both
the light and electron microscopical level.

Pre-embedding ISH

The pre-embedding in situ hybridization method as
presented here was used to localize a specific RNA
sequence in the nuclear matrix. The nuclear matrix is
defined as the structure in the nucleus that resists
treatment with strong detergents as present in the CSKbuffer
(Fey et al., 1986). For this approach the use of
CSK-buffer was therefore mandatory. For general
applications where the localization of nuclear or
cytoplasmic nucleic acid sequences is the objective, it
may not always be required to use such extreme
conditions. Much of the cytoplasmic and membranous
structure is removed as a consequence of this treatment.
This may not only hamper interpretation of the
ultrastructure but may also lead to removal of target
molecules. A limited degree of permeabilization will
however always be necessary with the pre-embedding
approach, if only to render the plasma membranes
permeable. Alternatives may be found in the use of
saponin (Willingham, 1983) which must be present
during all of the steps from fixation to the wash steps
after the immunogold incubation. For the
immunocytochemical localization of cytoskeletal
components it was found that even a sodium
borohydride treatment may be sufficient to allow ultra
small immunogold conjugates to pass the plasma
membrane of paraformaldehyde/glutaraldehyde fixed
neurones (van Lookeren Campagne, 1993) and in 0.5%
glutaraldehyde fixed epithelial cells (Leunissen and van
de Plas, personal communication). Finally, a
dehydration/rehydration sequence with graded ethanol
and acetone, as used in standard Epon-812 embedding,
may prove useful.

Post-embedding ISH

Similarity with pre-embedding

From the schedules presented on page 4 it follows that
the sequence of hybridization, protein
block/immunoincubation and enhancement/staining
steps in post-embedding in situ hybridization is not
different from the one used in pre-embedding. The
same holds for the pre-treatment and pre-hybridization
steps (if applied or necessary).

Differences with pre-embedding
Permeabilization vs. sectioning

The major difference is found in the omission of a
physicochemical permeabilization step based on the use
of detergents. Instead accessibility of the target is
achieved by making sections of the specimen.

Sections which can be fully rehydrated (paraffin,
cryostat, ultrathin cryosections) before proceeding with
the specific localization steps may have advantages
over sections of plastic embedded material on the
following points:

More targets are preserved;

The section surface is not as smooth as found with
plastic sections. This leads to higher detection
efficiencies at the section surface;

In the depth of the hydrated section more targets may
be exposed and available for binding with the nucleic
acid probe and the immunoconjugate.

The wash steps after the immunogold incubation may
be limited to a total of 30 minutes instead of 2 hours
since a limited reactive area is available.

DNA-ISH

With the following minor adaptations the presented
approach should also be useful to localize DNA
sequences:

An RNAse step has to be performed instead of the
DNAse step (temperature preferentially 37°C);

Furthermore the DNA present in the specimen has to
be denatured before hybridization with a nucleic acid
probe can take place. This can be achieved in two ways:
-- either through a pre-hybridization step using the
hybridization mixture without probe for 10 minutes at
80°C prior to the actual hybridization or
-- by incubating at 80°C with the complete
hybridization mixture for 10 minutes after which the
temperature is lowered to the hybridization
temperature.

Detection systems

The results obtained with the alkaline phosphatase
colour reaction and the detection with gold conjugates
are highly comparable. The detection method with the
gold conjugates as described here involves a one-stepdetection
method i.e. the hybrids are detected with an
anti-digoxigenin F(ab)-fragment directly coupled to
ultra small gold particles. The signal can be improved
with a two-step detection system using secondary
antibody gold conjugates. For instance the
digoxigeninylated nucleic acid can be detected with a
mouse monoclonal antibody which in turn is detected
with a goat-anti-mouse IgG ultra small gold conjugate.

For starters

When immunogold detection of nucleic acids is aimed
at, the following approach is recommended:
Firstly optimize the hybridization procedure in
combination with alkaline phosphatase detection at the
light microscopical level, since the detection with
alkaline phosphatase is easy and sensitive. The
hybridization conditions found to be optimal in this
system are also valid for the immunogold detectin
system. After establishing a specific hybridization
signal with immunogold detection at the light
microscopical level the samples can be prepared for
examination at the electron microscopical level.

View our digital catalog

Follow us on...

Electron Microscopy Sciences specializes in the manufacturing, preparation and distribution
of the highest quality laboratory chemicals and microscopy supplies and equipment
for electron microscopy, light microscopy, and histology.